Proteins specific for BAFF and B7RP1 and uses thereof

ABSTRACT

Described herein are bispecific proteins specific for BAFF and B7RP1, nucleic acids encoding such proteins, methods of making such proteins, and uses for such proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US Provisional Application Nos.61/780,260, filed Mar. 13, 2013, and 61/942,776, filed Feb. 21, 2014,each of which are incorporated herein in their entirety.

FIELD

The bispecific molecules described herein are within the field ofprotein therapeutics.

BACKGROUND

Most therapeutic proteins bind to a single target protein with highspecificity, thereby interfering with the activity of this single targetprotein. That protein may be a part of one or more biological pathwaysthat mediate a human disease being treated, and the therapeutic proteinmay therefore inhibit disease progression. However, efficacy oftherapeutic proteins is rarely complete for all patients. Incompleteefficacy of therapeutic proteins could be due in some cases to thecomplexity of a disease. For example, some diseases may be mediated bymultiple biological pathways, or different biological pathways may playa predominant role in mediating disease activity in different patientshaving the same clinically-defined condition. Hence, in some diseases itmay be advantageous to simultaneously inhibit at least two biologicalpathways.

SUMMARY

Herein is provided a bispecific protein that can bind to and inhibit thebiological activity of both human B7-related protein 1 (B7RP1, alsoknown as GL50 and T-cell co-stimulator ligand (ICOSLG)) and human B-cellactivating factor (BAFF, also known as tumor necrosis factorsuperfamily, member 13b (TNFSF13B)). BAFF plays a role in B cellsurvival, and B7RP1 plays a role in T cell costimulation. Thus, aprotein that inhibits the activity of both proteins interferes with theactivity of both B and T cells.

Described herein is bispecific protein, wherein the protein can inhibitBAFF-mediated proliferation of human B cells and wherein the protein caninhibit B7RP1-mediated proliferation of human T cells. The bispecificprotein can comprise an IgG antibody comprising two immunoglobulin heavychains having different amino acid sequences and two immunoglobulinlight chains having different amino acid sequences. The IgG antibody caninhibit BAFF-mediated proliferation of human B cells and B7RP1-mediatedproliferation of human T cells. The IgG antibody can be an IgG1, IgG2,IgG3, or IgG4 antibody and can be a human or humanized IgG antibody. Thebispecific protein can comprise a light chain complementaritydetermining region 1 (CDR1) comprising the amino acid sequence of SEQ IDNO:8, a light chain complementarity determining region 2 (CDR2)comprising the amino acid sequence of SEQ ID NO:9, a light chaincomplementarity determining region 3 (CDR3) comprising the amino acidsequence of SEQ ID NO:10, a heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO:11, a heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:12, and a heavy chain CDR3 comprising the aminoacid sequence of SEQ ID NO:13. Further, the bispecific protein cancomprise a heavy chain variable region comprising SEQ ID NO:15 or avariant thereof and a light chain variable region comprising SEQ IDNO:14 or a variant thereof. Such variant sequences can comprise not morethan 10 deletions, insertions of substitutions of a single amino acidper 100 amino acids relative to a reference sequence.

In an alternate embodiment, a bispecific protein that can inhibitBAFF-mediated proliferation of human B cells and that can inhibitB7RP1-mediated proliferation of human T cells can comprise: (a) apolypeptide comprising an amino acid sequence having the followingformula: A-L1-P-L2-P, wherein A is an immunoglobulin heavy chain of anIgG antibody, L1 is a first linker of that is absent or is 3 to 40 aminoacids long, P is a BAFF-binding peptide that is 10 to 40 amino acidslong, and L2 is a peptide linker that is absent or is 5 to 50 aminoacids long; and (b) an immunoglobulin light chain. The immunoglobulinheavy chain of (a) and the immunoglobulin light chain of (b) can form anIgG antibody, comprising two molecules of the polypeptide of (a) and twomolecules of the light chain of (b), that can bind B7RP1 and/or caninhibit B7RP1-mediated proliferation of human T cells. Theimmunoglobulin heavy chain may be missing a lysine at its C-terminal endjust upstream of L1. The IgG antibody can be a human or humanized IgG1,IgG2, IgG3, or IgG4 antibody. The BAFF-binding peptide P can have theamino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. L1 canhave the amino acid sequence of SEQ ID NO:4, 37, 38, 39, or 40. L2 canhave the amino acid sequence of SEQ ID NO:5, 6, or 7. The bispecificprotein can comprise a light chain CDR1 comprising the amino acidsequence of SEQ ID NO:8 (RASQGISNWLA), a light chain CDR2 comprising theamino acid sequence of SEQ ID NO:9 (AASSLQS), a light chain CDR3comprising the amino acid sequence of SEQ ID NO:10 (QQYDSYPRT), a heavychain CDR1 comprising the amino acid sequence of SEQ ID NO:11 (SYWMS), aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:12(YIKQDGNEKYYVDSVKG), and a heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO:13 (EGILWFGDLPTF). The bispecific protein cancomprise an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:14 and/or an immunoglobulin heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:15. Thebispecific protein can comprise the amino acid sequence of SEQ ID NO:19or a variant thereof and the amino acid sequence of SEQ ID NO:17 or 18or variants thereof. Such variant sequences can comprise not more than10 deletions, insertions of substitutions of a single amino acid per 100amino acids relative to the reference sequence.

In a further aspect, herein is described a bispecific proteincomprising: (a) a polypeptide comprising the amino acid sequence of SEQID NO:17 or SEQ ID NO:18 or variants thereof; and (b) anotherpolypeptide comprising the amino acid sequence of SEQ ID NO:19 or avariant thereof. Such variant sequences can comprise not more than 10deletions, insertions of substitutions of a single amino acid per 100amino acids relative to the reference sequence. The bispecific proteincan inhibit BAFF-mediated proliferation of human B cells andB7RP1-mediated proliferation of human T cells. The bispecific proteincan be a tetramer comprising two molecules of the polypeptide of (a) andtwo molecules of the polypeptide of (b).

In another embodiment, herein is provided a protein comprising a linkercomprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7. Insome embodiments, this protein can inhibit BAFF-mediated proliferationof human B cells and/or B7RP1-mediated proliferation of human T cells.Such a protein can comprise the amino acid sequences of SEQ ID NO:1, SEQID NO:14, and/or SEQ ID NO:15. Such a protein can comprise an amino acidsequence comprising at least two copies of SEQ ID NO:1 separated by SEQID NO:6 or 7. In a further embodiment, such a protein can include animmunoglobulin light chain and an immunoglobulin heavy, and SEQ ID NO:6or 7 can be downstream from the C-terminus of the heavy chain. In suchembodiments, SEQ ID NO:6 or 7 can be flanked by peptides that bind to aprotein other than that bound by the heavy and light chains.

Further, herein is described a pharmaceutical composition comprising anyof the bispecific proteins herein described or the protein comprisingthe amino acid sequence of SEQ ID NO:6 or 7 and a physiologicallyacceptable excipient.

Also described herein is a nucleic acid encoding any polypeptideincluded in one of bispecific proteins or the proteins comprising SEQ IDNO:6 or SEQ ID NO:7 herein described. Exemplary nucleic acids encoding apolypeptide included in a bispecific protein include, for example, SEQID NOs: 55, 56, 60, 61, 62, and 63, among others. Vectors comprisingsuch nucleic acids and host cells containing such vectors and/or nucleicacids are described. Further described herein is method for making abispecific protein comprising culturing the host cell containing anucleic acid encoding any of the bispecific proteins described hereinunder conditions such that the nucleic acid is expressed and recoveringthe protein from the cell mass or the culture medium. The host cell canbe a mammalian cell, for example, a CHO cell, or a bacterial cell suchas Eschericha coli.

In another aspect, described herein is a method for treating systemiclupus erythematosus, including lupus nephritis, comprising administeringto a patient a therapeutically effective amount of any of the bispecificproteins described herein or a pharmaceutical composition comprisingsuch a bispecific protein. Another therapeutic can be administered tothe patient before, after, or concurrently with the bispecific protein.The other therapeutic can be a corticosteroid, an antimalarial, retinoicacid, an NSAID, cyclophosphamide, dehydroepiandrosterone, mycophenolatemofetil, azathioprine, chlorambucil, methotrexate, tacrolimus, dapsone,thalidomide, leflunomide, or cyclosporine.

In a further aspect, herein is described a method of treatmentcomprising administering to a patient a therapeutically effective amountof any of the bispecific proteins described herein or a pharmaceuticalcomposition comprising a bispecific protein described herein, whereinthe patient has a disease selected from the group consisting of:ANCA-positive vasculitis, rheumatoid arthritis (RA), Crohn's disease,ulcerative colitis, celiac disease, pemphigus, pemphigoid, subacutecutaneous lupus erythematosus (SCLE), multiple sclerosis, chronicinflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis,Goodpasture's syndrome, glomerulonephritis, autoimmune hemolytic anemia(AIHA), idiopathic thrombocytopenic purpura (ITP), chronic activehepatitis, primary billiary cirrhosis, Sjogren's syndrome, systemicsclerosis, Hashimoto's thyroiditis, Graves' disease, Addison's disease,and multiple endocrine neoplasia (MEN).

In another aspect, herein is described a pharmaceutical compositioncomprising any of the bispecific proteins or the proteins comprising SEQID NO:6 or SEQ ID NO:7 herein described. The pharmaceutical compositioncan be, for example, for the treatment of systemic lupus erythematosusor lupus nephritis.

In another aspect, the use of any of the bispecific proteins providedherein as a medicament is described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Diagrams of bispecific proteins that bind to BAFF and B7RP1.Across the top row are listed the identifier for each construct. Acrossthe second row is a brief descriptive phrase relating to the structureof each construct. Across the bottom row is a diagram of the structureof each construct. The unfilled ovals represent constant regions of animmunoglobulin heavy or light chain. The ovals filled with horizontallines represent immunoglobulin heavy or light chain variable (VH or VL)regions. The small, solidly filled squares and loops representBAFF-binding peptides. The hinge regions are shown as heavy verticallines, while the disulfide bridges are shown as heavy horizontal lines.The sequence of “G4S” in FIG. 1 is disclosed in SEQ ID NO: 72.

FIGS. 2A and 2B: Activity of bispecific proteins in a human B cellproliferation assay. The data shown in FIGS. 2A (top) and 2B (bottom)are from B cell proliferation assays performed as described inExample 1. In both panels, the x axis indicates the concentration(log[nM]) of the bispecific protein contained in the assay mixture, andthe y axis indicates the amount of ³H-thymidine uptake (counts perminute (cpm)). The meaning of each symbol is indicated by an identifierfor each protein assayed. Meanings of the identifiers are shown in FIG.1 and explained in Example 1.

FIG. 3: Activity of bispecific proteins in a human T cell proliferationassay. The data shown is from T cell proliferation assays performed asdescribed in Example 1. The x axis indicates the concentration (log[nM])of the bispecific or αB7RP1 antibody in the assay mixture, and the yaxis indicates percent of T cell ³H-thymidine uptake in the presence ofB7RP1 inhibitors at the indicated concentrations relative to T cell³H-thymidine uptake without B7RP1 inhibitors (percent of control). Theidentifier for each protein tested is indicated.

FIG. 4: Cytokine release by human tonsil cells stimulated withStaphylococcus enterotoxin B (SEB). Methods are described in Example 1.The y axes show the levels of signal detected for the various cytokinesmeasured using Meso Scale Discovery (Rockville, Md.) kits according tothe manufacturer's instructions. The cells were treated with eitherαB7RP1 (lane 1), P74293 (lane 2), CTLA4-Ig (lane 3), or human IgG (lane4). The cytokines assayed are indicated in the figure.

FIG. 5: Pharmacokinetic profile of bispecific constructs in mice.Methods for assessing the in vivo pharmacokinetic properties of P71617,P71619, P71621, P71622, P74293, and P74294 in mice are described inExample 1. As explained in Example 1, the bispecific proteins weredetected by two different assays, one of which detected only the Fcportion of the proteins (data points indicated by filled diamonds; Fcassay) and one of which detected both the Fc and BAFF-binding portion ofthe proteins (data points indicated by filled squares; intact assay).The x axis indicates the time post injection (hours), and the y axisindicates the concentration of the protein detected in serum (ng/mL).The construct injected is indicated in each panel.

FIG. 6A: Inhibition of murine B cell proliferation by a murine surrogatebispecific molecule (the “murine surrogate”) that binds to BAFF andB7RP1. The assay was performed as described in Example 2. The murinesurrogate comprises an anti-murine B7RP1 IgG antibody that has twocopies of a BAFF-binding peptide attached to the C terminus of theimmunoglobulin heavy chain of the antibody, as explained in Example 2.The positive control was a BAFF-binding peptibody (“αBAFF”). Data fromthe murine surrogate and αBAFF are indicated, respectively, by solidlyfilled circles and squares. The x axis indicates the concentration ofthese test proteins in the assay (log[pM]), and the y axis indicates³H-thymidine incorporation (cpm).

FIG. 6B: Inhibition of B7RP1 binding to murine T cells by the murinesurrogate. The assay was performed as described in Example 2. Ananti-murine B7RP1 IgG antibody (“anti-mB7RP1”) was used as a positivecontrol. Data from the murine surrogate and anti-mB7RP1 are indicated,respectively, by solidly filled circles and squares. The x axisindicates the concentration of these test proteins in the assay(log[pM]), and the y axis indicates the percent of murine B7RP1-Fc boundto the T cells.

FIG. 7: In vivo effects on immunological parameters of administration ofsheep red blood cells to mice. All results shown in this figure are fromassays described in Example 2. The proteins that the mice were treatedwith are indicated by the fill in each bar as follows: unfilled,anti-mB7RP1; vertical lines, αBAFF; horizontal lines, anti-mB7RP1 plusαBAFF; diagonal lines, the murine surrogate; checkerboard, mIgG1; andsolid fill (in bottom panel only), mice not challenged with SBRC. Toppanel, percentage of spleen B cells in mice challenged with sheep redblood cells (SRBC). The y axis indicates the percent of cells from thespleen that are B cells. Middle panel, percentage of spleen CD4⁺ T cellsthat are memory T cells in mice challenged with SRBC. Bottom panel,levels of anti-SRBC antibodies in serum from mice challenged with SRBC.

FIG. 8A: Proteinuria in NZB/NZW mice treated with various proteins.Methods are described in Example 2. The treatment for each group of miceis indicated as follows: filled circles, phosphate buffered saline(PBS); filled squares, murine IgG1 (an isotype control; 5 mg/kg);unfilled squares, anti-mB7RP1 (4.68 mg/kg); filled, upward-pointingtriangles, αBAFF (1.88 mg/kg); unfilled, upward-pointing triangles,αBAFF (1.88 mg/kg) plus anti-mB7RP1 (4.68 mg/kg); and unfilled,downward-pointing triangles, the murine surrogate (5 mg/kg). The x axisindicates the age of the mice (months), and the y axis indicates thepercent of mice that exhibited proteinuria, i.e., ≥300 mb/dL protein inurine.

FIG. 8B: Levels of antibodies against double stranded DNA (dsDNA) inNZB/NZW mice at 8.5 months of age treated with various proteins. Methodsare described in Example 2. The x axis indicates the identity of themolecule(s) that the mice were treated with as follows: 1, anti-mB7RP1(4.68 mg/kg); 2, αBAFF (1.88 mg/kg); 3, αBAFF (1.88 mg/kg) plusanti-mB7RP1 (4.68 mg/kg); 4, the murine surrogate bispecific (5 mg/kg);and 5, mIgG1 (the isotype control; 5 mg/kg). The y axis indicates thelevels of anti-dsDNA antibodies detected as a percentage of the positivecontrol. Each dot indicates data from a single mouse.

FIG. 9A: Levels of anti-dsDNA IgG in NZB/NZW mice. Methods are describedin Example 2. Data from various groups of mice are identified asfollows: 1, mice that received anti-mB7RP1 (14 mg/kg); 2, mice thatreceived αBAFF (5.6 mg/kg); 3, mice that received a combination ofanti-mB7RP1 (14 mg/kg) and αBAFF (5.6 mg/kg); 4, mice that received themurine surrogate (15 mg/kg); 5, mice that received the mIgG isotypecontrol (15 mg/kg); and 6, mice that received PBS. The asterisks abovelanes 1, 3, and 4 indicate a significant (*, p<0.05; ***, p<0.0001)difference between data in those lanes and data from lane 5 (mIgG).

FIG. 9B: Percentages of NZB/W F₁ mice in each group having proteinuria.Methods are described in Example 2. Data from various groups of mice areidentified as follows: unfilled squares, mice that received anti-mB7RP1(14 mg/kg); filled, upward-pointing triangles, mice that received αBAFF(5.6 mg/kg); unfilled, upward-pointing triangles, mice that received acombination of anti-mB7RP1 (14 mg/kg) and αBAFF (5.6 mg/kg); unfilled,downward-pointing triangles, mice that received the murine surrogate (15mg/kg); filled squares, mice that received the mIgG isotype control (15mg/kg); and filled circles, mice that received PBS. Significantdifferences were detected between the murine surrogate versusanti-mB7RP1 (p<0.01), αBAFF (p<0.0001), and mIgG (p<0.0001). The timewindow in which treatment occurred is indicated.

FIG. 10: Kidney scores of NZB/W F₁ mice. As explained in Example 2,kidneys were harvested when a mouse died, if that happened before theend of the study, or at the end of the study. Kidney scores weredetermined as described in Example 2, with higher scores indicating moresevere kidney disease. Shown are averages for each group of mice plusappropriate error bars. The groups of mice received the followingtreatments: 1) anti-mB7RP1 (14 mg/kg), bar filled with vertical lines;2) αBAFF (5.6 mg/kg), bar filled with horizontal lines; 3) combinationof anti-mB7RP1 (14 mg/kg) and αBAFF (5.6 mg/kg), bar filled withwindowpane checks; 4) the murine surrogate (15 mg/kg), bar filled withcheckerboard pattern; 5) mIgG (15 mg/kg), bar filled with white dots ona black background; and 6) PBS, solidly filled bar. Asterisks indicate asignificant difference from mice treated with mIgG with a p value of<0.05 (*) or <0.001 (***).

FIG. 11: Effects of inhibition of BAFF and/or B7RP1 on murinecollagen-induced arthritis. Methods are described in Example 4. The fivegroups of mice were treated with test substances indicated as follows:mIgG, filled squares connected by solid lines; PBS, filled squaresconnected by dashed lines; anti-mB7RP1, filled circles connected bydashed lines; αBAFF, open circles connected by solid lines; andcombination of anti-mB7RP1 and αBAFF, filled circles connected by solidlines. The top panel shows the percent incidence of arthritis of thevarious groups, and the bottom panel shows the average arthritic scoresof the groups. The vertical, downward-pointing arrow in each panelindicates the time of the second immunization with bovine collagen.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS SEQUENCE LISTING NUMBERDESCRIPTION SEQ ID NO: 1 Amino acid sequence of a BAFF-binding peptideSEQ ID NO: 2 Amino acid sequence of a BAFF-binding peptide SEQ ID NO: 3Amino acid sequence of a BAFF-binding peptide SEQ ID NO: 4 Amino acidsequence of a linker SEQ ID NO: 5 Amino acid sequence of a linker SEQ IDNO: 6 Amino acid sequence of a linker SEQ ID NO: 7 Amino acid sequenceof a linker SEQ ID NO: 8 Amino acid sequence of a light chain CDR1 SEQID NO: 9 Amino acid sequence of a light chain CDR2 SEQ ID NO: 10 Aminoacid sequence of a light chain CDR3 SEQ ID NO: 11 Amino acid sequence ofa heavy chain CDR1 SEQ ID NO: 12 Amino acid sequence of a heavy chainCDR2 SEQ ID NO: 13 Amino acid sequence of a heavy chain CDR3 SEQ ID NO:14 Amino acid sequence of a light chain variable region SEQ ID NO: 15Amino acid sequence of a heavy chain variable region SEQ ID NO: 16 Aminoacid sequence of a heavy chain of the P71619 BAFF/B7RP1 bispecificmolecule SEQ ID NO: 17 Amino acid sequence of a heavy chain of theP74293 BAFF/B7RP1 bispecific molecule SEQ ID NO: 18 Amino acid sequenceof a heavy chain of the P74294 BAFF/B7RP1 bispecific molecule SEQ ID NO:19 Amino acid sequence of the immunoglobulin light chain of an IgGanti-huB7RP1 antibody SEQ ID NO: 20 Amino acid sequence preceding aheavy chain CDR1 SEQ ID NO: 21 Amino acid sequence preceding a heavychain CDR2 SEQ ID NO: 22 Amino acid sequence following heavy chain CDR3SEQ ID NO: 23 Amino acid sequence following light chain CDR3 SEQ ID NO:24 Linker SEQ ID NO: 25 Amino acid sequence of the immunoglobulin heavychain of an anti-B7RP1 IgG antibody SEQ ID NO: 26 Amino acid sequence ofheavy chain of construct P71617 SEQ ID NO: 27 Amino acid sequence oflight chain of construct P71618 SEQ ID NO: 28 Amino acid sequence ofheavy chain of construct P71620 SEQ ID NO: 29 Amino acid sequence of theheavy chain of the P71621 construct SEQ ID NO: 30 Amino acid sequence ofthe heavy chain of construct P71622 SEQ ID NO: 31 Amino acid sequence ofthe heavy chain of construct P71623 SEQ ID NO: 32 Amino acid sequence ofαBAFF peptibody SEQ ID NO: 33 Amino acid sequence of human IgG1 Fcregion SEQ ID NO: 34 Amino acid sequence of human IgG2 Fc region SEQ IDNO: 35 Amino acid sequence of human IgG3 Fc region SEQ ID NO: 36 Aminoacid sequence of human IgG4 Fc region SEQ ID NO: 37 Amino acid sequenceof a linker SEQ ID NO: 38 Amino acid sequence of a linker SEQ ID NO: 39Amino acid sequence of a linker SEQ ID NO: 40 Amino acid sequence of alinker SEQ ID NO: 41 Nucleic acid sequence encoding the amino acidsequence of SEQ. ID NO: 1 SEQ ID NO: 42 Nucleic acid sequence encodingthe amino acid sequence of SEQ ID NO: 4 SEQ ID NO: 43 Nucleic acidsequence encoding the amino acid sequence of SEQ ID NO: 5 SEQ ID NO: 44Nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6SEQ ID NO: 45 Nucleic acid sequence encoding the amino acid sequence ofSEQ ID NO: 7 SEQ ID NO: 46 Nucleic acid sequence encoding the amino acidsequence of SEQ ID NO: 8 SEQ ID NO: 47 Nucleic acid sequence encodingthe amino acid sequence of SEQ ID NO: 9 SEQ ID NO: 48 Nucleic acidsequence encoding the amino acid sequence of SEQ ID NO: 10 SEQ ID NO: 49Nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11SEQ ID NO: 50 Nucleic acid sequence encoding the amino acid sequence ofSEQ ID NO: 12 SEQ ID NO: 51 Nucleic acid sequence encoding the aminoacid sequence of SEQ ID NO: 13 SEQ ID NO: 52 Nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO: 14 SEQ ID NO: 53 Nucleicacid sequence encoding the amino acid sequence of SEQ ID NO: 15 SEQ IDNO: 54 Nucleic acid sequence encoding the amino acid sequence of SEQ IDNO: 16 SEQ ID NO: 55 Nucleic acid sequence encoding the amino acidsequence of SEQ ID NO: 17 SEQ ID NO: 56 Nucleic acid sequence encodingthe amino acid sequence of SEQ ID NO: 18 SEQ ID NO: 57 Nucleic acidsequence encoding the amino acid sequence of SEQ ID NO: 19 SEQ ID NO: 58Nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24SEQ ID NO: 59 Nucleic acid sequence encoding the amino acid sequence ofSEQ ID NO: 25 SEQ ID NO: 60 Nucleic acid sequence encoding the aminoacid sequence of SEQ ID NO: 26 SEQ ID NO: 61 Nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO: 27 SEQ ID NO: 62 Nucleicacid sequence encoding the amino acid sequence of SEQ ID NO: 28 SEQ IDNO: 63 Nucleic acid sequence encoding the amino acid sequence of SEQ IDNO: 29 SEQ ID NO: 64 Nucleic acid sequence encoding the amino acidsequence of SEQ ID NO: 30 SEQ ID NO: 65 Nucleic acid sequence encodingthe amino acid sequence of SEQ ID NO: 31 SEQ ID NO: 66 Nucleic acidsequence encoding the amino acid sequence of SEQ ID NO: 32 SEQ ID NO: 67Nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 33SEQ ID NO: 68 Nucleic acid sequence encoding the amino acid sequence ofSEQ ID NO: 34 SEQ ID NO: 69 Nucleic acid sequence encoding the aminoacid sequence of SEQ ID NO: 35 SEQ ID NO: 70 Nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO: 36 SEQ ID NO: 71 Aminoacid sequence of a linker SEQ ID NO: 72 Amino acid sequence of a linker

DETAILED DESCRIPTION

Provided herein are bispecific proteins that bind to and inhibit both Bcell activating factor (BAFF; also known as BLYS, TAL1, THANK, orTNFSF13B) and B7-related protein 1 (B7RP1; also known as ICOS Ligand,ICOSL, LICOS, B7 Homolog 2, B7H2, and GL50), nucleic acids encodingthese bispecific proteins, and methods of making and using theseproteins. The bispecific proteins can inhibit both BAFF-mediated Bproliferation and B7RP1-mediated T cell proliferation. In anotheraspect, the bispecific proteins can inhibit B7RP1 binding to T cells.Such a bispecific protein can be an IgG antibody comprising twodifferent immunoglobulin heavy chains and two different immunoglobulinlight chains, where one heavy chain/light chain pair binds to BAFF andthe other binds to B7RP1. Alternatively, the B7RP1-binding portion ofthe bispecific protein can comprise an IgG antibody including twoidentical heavy chains and two identical light chains, and theBAFF-binding portion of the bispecific protein can comprise one or moreBAFF-binding peptides, which can be fused to the anti-B7RP1 antibody,optionally via the N-terminus of the immunoglobulin heavy or lightchain, the carboxyterminus of the immunoglobulin heavy chain, and/orwithin the CH2 and/or CH3 region of the immunoglobulin heavy chain.

Definitions

An “antibody,” as meant herein, is a protein comprising a heavy and/orlight chain immunoglobulin variable region.

A “bispecific” protein, as meant herein is a protein that can bindspecifically to two different molecules, which, in some embodiments, areproteins. For example, in some embodiments, a bispecific protein canbind to both BAFF and B7RP1.

A patient is receiving “concurrent” treatment with two or moretherapeutics when the patient receives the two or more therapeuticsduring the same general timeframe, optionally at the very same time. Forexample, if a patient were dosed with one therapeutic daily on anongoing basis and were also dosed with another therapeutic once a monthon an ongoing basis, the patient would be receiving these two drugsconcurrently. Similarly, a patient dosed with two differenttherapeutics, each administered every two weeks, but not on the sameday, would be receiving concurrent treatment with the two therapeutics.Further, a patient receiving one therapeutic on an ongoing basis onceper week and another therapeutic once per day for only three days wouldbe receiving treatment for a short period of time with these twotherapeutics.

As meant herein, an “Fc region” is a dimer consisting of two polypeptidechains joined by one or more disulfide bonds, each chain comprising partor all of a hinge domain plus a CH2 and a CH3 domain. Each of thepolypeptide chains is referred to as an “Fc polypeptide chain.” Morespecifically, the Fc regions contemplated for use with the presentinvention are IgG Fc regions, which can be mammalian, for example human,IgG1, IgG2, IgG3, or IgG4 Fc regions. Among human IgG1 Fc regions, atleast two allelic types are known. The amino acid sequences an Fcpolypeptide chain can vary from those of a mammalian Fc polypeptide byno more than 20, 15, 12, 10, 8, 5, or 3 substitutions, insertions, ordeletions of a single amino acid relative to a mammalian Fc polypeptideamino acid sequence. Alternatively or in addition, the amino acidsequence of an Fc polypeptide chain can vary from the sequence of aknown or naturally occurring Fc polypeptide chain by no more thant 10insertions, deletions, or substitutions of a single amino acid per every100 amino acids of sequence. In some embodiments, such variations can be“heterodimerizing alterations” that facilitate the formation ofheterodimers over homodimers. In referring to particular positionswithin an Fc polypeptide chain, the EU numbering system (Edelman et al.(1969), Proc. Natl. Acad. Sci. 63: 78-85) is used, as illustrated in thealignment of human IgG Fc polypeptide chains in Table 1 below.

TABLE 1 Alignment of amino acid sequences of human IgG Fc regions IgG1             ----------------------------------------------- IgG2             ----------------------------------------------- IgG3             ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP IgG4             -----------------------------------------------        225       235       245       255       265       275         *         *         *         *         *         * IgG1EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF IgG2ERKCCVE---CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG3EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF IgG4ESKYG---PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF        285       295       305       315       325       335         *         *         *         *         *         * IgG1NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG2NWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT IgG3KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG4NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT        345       355       365       375       385       395         *         *         *         *         *         * IgG1ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG2ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG3ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP IgG4ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP        405       415       425       435       445         *         *         *         *         * IgG1PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33)IgG2PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34)IgG3PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 35)IgG4PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 36)At some positions, naturally-occurring polymorphisms can occur. Forexample, the methionine at position 282 in the IgG2 sequence given aboveis more typically a valine in naturally occurring IgG2 sequences.Similarly, the tyrosine at position 296 in an IgG3 sequence can also bea phenylalanine.

“Heterodimerizing alterations” generally refer to alterations in the CH3regions two different IgG heavy chains that facilitate the formation ofheterodimeric heavy chain dimers, that is, dimerized heavy chains thatdo not have identical amino acid sequences. Heterodimerizing alterationscan be asymmetric, that is, one heavy chain having a certain alterationcan pair with another heavy chain having a different alteration. Thesealterations facilitate heterodimerization and disfavor homodimerization.One example of such paired heterodimerizing alterations are theso-called “knobs and holes” substitutions. See, e.g., U.S. Pat. No.7,695,936 and US Patent Application Publication 2003/0078385, theportions of which describe such mutations are incorporated herein byreference. As meant herein, heavy chain-heavy chain pair that containsone pair of knobs and holes substitutions, contains one substitution inone heavy chain and another substitution in the other heavy chain. Forexample, the following knobs and holes substitutions have been found toincrease heterodimer formation as compared with that found withunmodified heavy chains: 1) Y407T in one chain and T366Y in the other;2) Y407A in one chain and T366W in the other; 3) F405A in one chain andT394W in the other; 4) F405W in one chain and T394S in the other; 5)Y407T in one chain and T366Y in the other; 6) T366Y and F405A in onechain and T394W and Y407T in the other; 7) T366W and F405W in one chainand T394S and Y407A in the other; 8) F405W and Y407A in one chain andT366W and T394S in the other; and 9) T366W in one polypeptide of the Fcand T366S, L368A, and Y407V in the other. As meant herein, mutations inan Fc polypeptide are denoted in the following way. The amino acid(using the one letter code) normally present at a given position in theCH3 region using the EU numbering system (which is presented in Edelmanet al. (1969), Proc. Natl. Acad. Sci. 63: 78-85) is followed by the EUposition number, which is followed by the alternate amino acid that ispresent at that position. For example, Y407T means that the tyrosinenormally present at EU position 407 is replaced by a threonine. For thesake of clarity, the EU system of numbering is illustrated in Table 1below. Alternatively or in addition to such alterations, substitutionscreating new disulfide bridges can facilitate heterodimer formation.See, e.g., US Patent Application Publication 2003/0078385, the portionsof which describe such mutations are incorporated herein by reference.Such alterations in an IgG1 Fc region include, for example, thefollowing substitutions: Y349C in one Fc-polypeptide chain and S354C inthe other; Y349C in one Fc-polypeptide chain and E356C in the other;Y349C in one Fc-polypeptide chain and E357C in the other; L351C in oneFc-polypeptide chain and S354C in the other; T394C in one Fc-polypeptidechain and E397C in the other; or D399C in one Fc-polypeptide chain andK392C in the other. Similarly, substitutions changing the charge of aone or more residue, for example, in the CH3-CH3 interface, can enhanceheterodimer formation as explained in WO 2009/089004, the portions ofwhich describe such substitutions are incorporated herein by reference.Such substitutions are referred to herein as “charge pairsubstitutions,” and an Fc region containing one pair of charge pairsubstitutions contains one substitution in one heavy chain and adifferent substitution in the other. General examples of charge pairsubstitutions include the following: 1) R409D, R409E, K409D, or K409E inone chain plus D399K or D399R in the other; 2) N392D, N392E, K392D, orK392E in one chain plus D399K or D399R in the other; 3) K439D or K439Ein one chain plus E356K, E356R, D356K, or D356R in the other; and 4)K370D or K370E in one chain plus E357K or E357R in the other. Inaddition, the substitutions Q355D, Q355E, R355D, R355E, K360D, or K360Rin both chains can stabilize heterodimers when used with otherheterodimerizing alterations. Specific charge pair substitutions can beused either alone or with other charge pair substitutions. Specificexamples of single pairs of charge pair substitutions and combinationsthereof include the following: 1) K409E in one chain plus D399K in theother; 2) K409E in one chain plus D399R in the other; 3) K409D in onechain plus D399K in the other; 4) K409D in one chain plus D399R in theother; 5) K392E in one chain plus D399R in the other; 6) K392E in onechain plus D399K in the other; 7) K392D in one chain plus D399R in theother; 8) K392D in one chain plus D399K in the other; 9) K409D and K360Din one chain plus D399K and E356K in the other; 10) K409D and K370D inone chain plus D399K and E357K in the other; 11) K409D and K392D in onechain plus D399K, E356K, and E357K in the other; 12) K409D and K392D onone chain and D399K on the other; 13) K409D and K392D on one chain plusD399K and E356K on the other; 14) K409D and K392D on one chain plusD399K and D357K on the other; 15) K409D and K370D on one chain plusD399K and D357K on the other; 16) D399K on one chain plus K409D andK360D on the other; and 17) K409D and K439D on one chain plus D399K andE356K on the other. Any of these heterodimerizing alterations can bepart of an immunoglobulin IgG heavy chain as described herein.

A “human” antibody or protein, as meant herein, is an antibody orprotein encoded by a nucleic acid sequence of human origin. A humanantibody or protein can be made in cultured non-human cells or in vivoin a transgenic organism into which a nucleic acid molecule encoding thehuman antibody or protein has been introduced. Alternatively, a humanantibody or protein can be made in cultured human cells or in a human invivo.

An “IgG antibody,” as meant herein, is an antibody that consistsessentially of the immunoglobulin domains present in mostnaturally-occurring IgG antibodies, i.e., a immunoglobulin heavy chaincomprising a heavy chain variable (VH) region, a first heavy chainconstant (CH1) region, a hinge region, a second heavy chain constant(CH2) region, and a third heavy chain constant (CH3) region and a lightchain comprising a light chain variable (VL) region and a light chainconstant (CL) region. Numerous sequences of such immunoglobulin domainsare reported throughout the scientific literature, e.g., in SEQUENCES OFIMMUNOLOGICAL INTEREST, Public Health Service, N.I.H., Bethesda, Md.,1991. An IgG antibody, as meant herein, is a tetramer consistingessentially of two heavy chains and two light chains.Naturally-occurring antibodies including only two immunoglobulin heavychains and no immunoglobulin light chains, such as some found in camelsand sharks (see, e.g., Muyldermans et al., 2001, J. Biotechnol.74:277-302; Desmyter et al., 2001, J. Biol. Chem. 276:26285-90;Streltsov et al. (2005), Protein Science 14: 2901-2909), are not “IgGantibodies” as meant herein. An IgG antibody can be human or can be fromanother species. In addition, an IgG antibody can contain no more than40, 35, 30, 25, 20, 15, 10, or 5 substitutions, insertions, and/ordeletions of a single amino acid relative to the amino acid sequence ofthe heavy or light chains of a naturally occurring IgG antibody.

An “immunoglobulin heavy chain” refers to a heavy chain of an IgG, IgA,IgM, IgE, or IgD antibody or variants thereof containing not more than40, 30, 25, 20, 15, 10, or 5 insertions, deletions, or substitutions ofa single amino acid relative to an immunoglobulin heavy chain encoded bynucleic acid sequences originating in nature. An “immunoglobulin IgGheavy chain” is limited to heavy chains from IgG antibodies or variantsthereof containing not more than 40, 30, 25, 20, 15, 10, or 5insertions, deletions, or substitutions of a single amino acid relativeto the amino acid sequence of an IgG heavy chain encoded by nucleic acidsequences originating in nature. An immunoglobulin heavy chain consistsessentially of a number of distinct regions or domains including a VHregion, a CH1 region, a hinge region, a CH2 region, and a CH3 region. Insome other isotypes, i.e., IgM and IgA, additional regions are includeddownstream from the CH3 region. Immunoglobulin heavy chains and theregions included therein are generally described in, e.g.,Carayannopoulos and Capra, Immunoglobulins: Structure and Function, pp.283-314 in FUNDAMENTAL IMMUNOLOGY, 3^(rd) Ed, Paul, ed., Raven Press,New York, 1993, which is incorporated herein by reference. In addition,numerous sequences of subregions of immunoglobulin heavy chains areknown in the art. See, e.g., Kabat et al., SEQUENCES OF PROTEINS OFIMMUNOLOGICAL INTEREST, Public Health Service N.I.H., Bethesda, Md.,1991. In some cases, a polypeptide chain that includes an immunoglobulinheavy chain plus some non-immunoglobulin sequences will be referred toherein as a “heavy chain.”

An “immunoglobulin light chain,” as meant herein, is a kappa or a lambdachain from a human antibody or an antibody from another species. Alsoincluded among immunoglobulin light chains, as meant herein, areproteins with no more than 20, 15, 10, or 5 insertions, deletions,and/or substitutions of a single amino acid relative to animmunoglobulin light chain encoded by nucleic acid sequences of naturalorigin. Immunoglobulin light chains are generally described in, e.g.,Carayannopoulos and Capra, Immunoglobulins: Structure and Function, pp.283-314 in FUNDAMENTAL IMMUNOLOGY, 3^(rd) Ed, Paul, ed., Raven Press,New York, 1993, which is incorporated herein by reference. Aimmunoglobulin light chain contains a VL region and a CL region.Numerous sequences of these regions are known in the art. See, e.g.,Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, PublicHealth Service N.I.H., Bethesda, Md., 1991. In some cases, a polypeptidechain that includes an immunoglobulin light chain plus somenon-immunoglobulin sequences will be referred to herein as a “lightchain.”

An “immunoglobulin variable region,” as meant herein, is a VH or VLregion, which can be of human origin or from another species.Immunoglobulin variable regions are generally described in, e.g.,Carayannopoulos and Capra, Immunoglobulins: Structure and Function, pp.283-314 in FUNDAMENTAL IMMUNOLOGY, 3^(rd) Ed, Paul, ed., Raven Press,New York, 1993, which is incorporated herein by reference. Also includedamong immunoglobulin variable regions, as meant herein, are proteinswith no more than 20, 15, 10, or 5 insertions, deletions, and/orsubstitutions of a single amino acid relative to an immunoglobulinvariable region encoded by nucleic acid sequences of natural origin. Animmunoglobulin variable region contains three hypervariable regions,known as complementarity determining region 1 (CDR1), complementaritydetermining region 2 (CDR2), and complementarity determining region 3(CDR3). These regions form the antigen binding site of an antibody. TheCDRs are embedded within the less variable framework regions (FR1-FR4).The order of these subregions within a variable region is as follows:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Numerous sequences of immunoglobulinvariable regions are known in the art. See, e.g., Kabat et al, SEQUENCESOF PROTEINS OF IMMUNOLOGICAL INTEREST, Public Health Service N.I.H.,Bethesda, Md., 1991.

CDRs can be located in a VH region sequence in the following way. CDR1starts at approximately residue 31 of the mature VH region and isusually about 5-7 amino acids long, and it is almost always preceded bya Cys-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO: 20) (where “Xxx” isany amino acid). The residue following the heavy chain CDR1 is almostalways a tryptophan, often a Trp-Val, a Trp-Ile, or a Trp-Ala. Fourteenamino acids are almost always between the last residue in CDR1 and thefirst in CDR2, and CDR2 typically contains 16 to 19 amino acids. CDR2may be immediately preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 21) andmay be immediately followed byLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. Other amino acids mayprecede or follow CDR2. Thirty two amino acids are almost always betweenthe last residue in CDR2 and the first in CDR3, and CDR3 can be fromabout 3 to 25 residues long. A Cys-Xxx-Xxx almost always immediatelyprecedes CDR3, and a Trp-Gly-Xxx-Gly (SEQ ID NO: 22) almost alwaysfollows CDR3.

Light chain CDRs can be located in a VL region in the following way.CDR1 starts at approximately residue 24 of the mature antibody and isusually about 10 to 17 residues long. It is almost always preceded by aCys. There are almost always 15 amino acids between the last residue ofCDR1 and the first residue of CDR2, and CDR2 is almost always 7 residueslong. CDR2 is typically preceded by Ile-Tyr, Val-Tyr, Ile-Lys, orIle-Phe. There are almost always 32 residues between CDR2 and CDR3, andCDR3 is usually about 7 to 10 amino acids long. CDR3 is almost alwayspreceded by Cys and usually followed by Phe-Gly-Xxx-Gly (SEQ ID NO: 23).

A “linker,” as meant herein, is a peptide that links two polypeptides. Alinker can be from 1-80 amino acids in length. In some embodiments, alinker can be 2-40, 3-30, or 3-20 amino acids long. In some embodiments,a linker can be a peptide no more than 14, 13, 12, 11, 10, 9, 8, 7, 6,or 5 amino acids long. In other embodiments, a linker can be 5-25, 5-15,10-20, or 20-30 amino acids long. In other embodiments, a linker can beabout, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In manycases, linkers lack free cysteine residues (i.e. and are therefore notinvolved in disulfide bonds) and also do not contain N-glycosylationsites (that is, Asn-Xxx-Ser/Thr, where X can be any amino acid exceptproline).

A “peptibody,” as meant herein, is one or more biologically activepeptides fused to an Fc region. Shimamoto et al. (2012), mAbs 4(5):586-591, the portions of which explain the structure of a peptibody andhow to make it are incorporated herein by reference.

A “peptide,” as meant herein, is a polypeptide that consists of a shortamino acid sequence, which may or may not be glycosylated and/or containmodified amino acids. A peptide can be from 2 to 75 amino acids long. Insome embodiments, a peptide is 3-60, 3-50, 3-40, 3-30, or 3-20 aminoacids long. In other embodiments, a peptide can be 5-25, 5-15, 10-20, or20-30 amino acids long. In other embodiments, a peptide can be about, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 amino acids long.

A “therapeutically effective amount” of a drug used to treat a diseaseis an amount that can reduce the severity of a disease, reduce theseverity of one or more symptoms associated with the disease or itstreatment, or delay the onset of more serious symptoms or a more seriousdisease that can occur with some frequency following the treatedcondition.

“Treatment” of any disease mentioned herein encompasses an alleviationof at least one symptom of the disease, a reduction in the severity ofthe disease, or the delay or prevention of disease progression to moreserious symptoms that may, in some cases, accompany the disease or leadto at least one other disease. Treatment need not mean that the diseaseis totally cured. A useful therapeutic agent needs only to reduce theseverity of a disease, reduce the severity of one or more symptomsassociated with the disease or its treatment, or delay the onset of moreserious symptoms or a more serious disease that can occur with somefrequency following the treated condition. For example, if the diseasewere an inflammatory bowel disease, a therapeutic agent used as atreatment may reduce the number of distinct sites of inflammation in thegut or the total extent of the gut affected. It may reduce pain and/orswelling, reduce symptoms such as diarrhea, constipation, or vomiting,and/or prevent perforation of the gut. A patient's condition can beassessed by standard techniques such as an x-ray performed following abarium enema or enteroclysis, endoscopy, colonoscopy, and/or a biopsy.Suitable procedures vary according to the patient's condition andsymptoms. Similarly, if the disease treated were systemic lupuserythematosus (SLE), disease activity could be evaluated using theSLEDAI index for scoring, as explained below.

Bispecific Proteins that Bind to BAFF and B7RP1

Disclosed herein are bispecific proteins that bind to B7RP1 and BAFFand/or that can inhibit B7RP1-mediated T cell proliferation andBAFF-mediated B cell proliferation in vitro. The BAFF and B7RP1 proteinsto which a bispecific protein as described herein binds can be humanproteins and/or can be proteins from another species such as cynomolgusmonkey, rhesus monkey, chimpanzee, mouse, and/or rabbit, among others.In some embodiments, a bispecific protein as described herein can, forexample, bind to both human (Homo sapiens) and cynomolgus monkey (Macacafascicularis) B7RP1 and BAFF proteins.

In some embodiments, these bispecific proteins can be bispecific IgGantibodies in which the B7RP1-binding portion and the BAFF-bindingportion each consists essentially of an immunoglobulin IgG heavy chainand an immunoglobulin light chain. Thus, such a bispecific antibodycontains two different immunoglobulin heavy chains and two differentimmunoglobulin light chains. Together, these two pairs of immunoglobulinheavy and light chains form a complete bispecific IgG antibody.Bispecific IgG antibodies are known in the art, and a number of otherformats for bispecific antibodies are also known. See, e.g., Kontermann,Bispecific Antibodies: Developments and Current Perspectives, pp. 1-28in BISPECIFIC ANTIBODIES, Kontermann, ed., Springer-Verlag, Berlin,Heidelburg, 2011, the portions of which describe these antibodies areincorporated herein by reference. Antibodies that can bind to BAFF andB7RP1, regardless of format, are contemplated herein. Bispecific IgGantibodies can be human, humanized, or chimeric and can be of the IgG1,IgG2, IgG3, or IgG4 isotype. In some embodiments, bispecific IgGantibodies can be conjugated to other moieties. Amino acid sequences ofanti-BAFF and anti-B7RP1 antibodies are known in the art. See e.g., U.S.Pat. No. 7,737,111 and U.S. Patent Application Publication US2011/0117093. The portions of these documents that describe suchantibodies are incorporated herein by reference. In some embodiments,such bispecific antibodies can comprise “heterodimerizing alterations,”as defined above, including charge pair substitutions, that facilitateformation of a heterotetrameric bispecific IgG antibody.

In other embodiments, the bispecific proteins described herein can befusion proteins comprising an antibody that binds to B7RP1, whichcomprises an immunoglobulin IgG heavy chain and an immunoglobulin lightchain, and a peptide that binds to BAFF. The BAFF-binding peptide can bepresent in one or multiple copies, such as two, three, four, five, six,seven, eight, or up to 16 copies. The BAFF-binding peptide may bind toBAFF proteins from species such as mouse, cynomolgus monkey, and/orhumans, among many other possible species. The antibody can be ananti-B7RP1 IgG antibody, optionally a human or humanized antibody thatbinds to human and/or cynomolgus monkey B7RP1. In some embodiments, alinker can be attached to the C terminus of the heavy chain of theanti-B7RP1 IgG antibody, followed by a first BAFF-binding peptide,another linker, and a second BAFF-binding peptide. A third, fourth,fifth, sixth, seventh, eighth, or up to sixteenth BAFF-binding peptidecan follow these two, optionally interspersed with linkers.Alternatively or in addition, one, two, three, four, five, six, seven,or eight BAFF-binding peptides can be inserted elsewhere in theanti-B7RP1 antibody, for example at the N terminus of the immunoglobulinheavy chain or immunoglobulin light chain or in a loop region in the CH2or CH3 region. The IgG antibody can be a mammalian antibody, such as ahuman or murine antibody. The anti-B7RP1 antibody can be a human orhumanized IgG1, IgG2, IgG3, or IgG4 antibody. In such bispecific fusionproteins comprising an anti-B7RP1 IgG antibody, the bispecific proteincan comprise a heavy chain comprising the amino acid sequence of SEQ IDNO:17 or SEQ ID NO:18 and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO:19. Variants comprising a heavy chainhaving an amino acid sequence containing no more than 30, 25, 20, 15,10, 5, or 3 insertions, deletions, or substitutions of a single aminoacid relative to SEQ ID NO: 17 or 18 are contemplated. Similarly,variants comprising an immunoglobulin light chain having an amino acidsequence containing no more 20, 15, 10, 8, 7, 5, or 3 insertions,deletions, or substitutions or a single amino acid relative SEQ ID NO:19are contemplated. Such bispecific proteins can be tetramers comprisingtwo polypeptides comprising the amino acid sequence of SEQ ID NO:17 or18 or a variant thereof and two light chains comprising the amino acidsequence of SEQ ID NO:19 or a variant thereof.

A BAFF-binding peptide portion of a bispecific fusion protein asdescribed above can comprise the amino acid sequence of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3. Such BAFF-binding peptides are described inU.S. Pat. No. 7,737,111, the relevant portions of which are incorporatedherein by reference. In some embodiments, there may be one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or sixteen copies of such a BAFF-binding peptidepresent in the bispecific protein. A BAFF-binding peptide can beattached to the carboxy end of the anti-B7RP1 antibody, for example, viaa linker. For example, the carboxy end of an anti-B7RP1 IgG antibody canbe followed by a linker having, for example, the amino acid sequence ofGly-Gly-Gly-Gly (SEQ ID NO:4). Examples of other suitable linkersinclude Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO:37),Gly-Gly-Gly-Pro (SEQ ID NO:38), Gly-Gly-Gly-Gln (SEQ ID NO:39), andGly-Gly-Gly-Gly-Gly (SEQ ID NO:40), among many others. This linker canbe followed by a BAFF-binding peptide. The BAFF-binding peptide can befollowed by another linker comprising, for example, the amino acidsequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:24.Other linker could also be used. This linker can be followed by anotherBAFF-binding peptide comprising, for example, the amino acid sequence ofSEQ ID NO:1.

In the bispecific fusion proteins described immediately above or in thebispecific heterotetrameric IgG antibodies described above, a VL regioncan contain a CDR1, a CDR2, and a CDR3 comprising the amino acidsequences of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. AVH region CDR1, CDR2, and CDR3 can comprise the amino acid sequences ofSEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. In someembodiments, a VL region of the IgG antibody can comprise the amino acidsequence of SEQ ID NO:14 or a variant thereof, and the VH region cancomprise the amino acid sequence of SEQ ID NO:15 or a variant thereof.Such variant sequences can comprise not more than 10 deletions,insertions of substitutions of a single amino acid per 100 amino acidsrelative to a reference sequence.

Proteins Comprising a Linker

Provided herein are linkers having the amino acid sequences of SEQ IDNO:5, 6, or 7 that confer favorable physical properties on a proteinthat contains them. As shown in Example 1 below, the use of twoparticular linkers, i.e., those having the amino acid sequences of SEQID NO:6 and SEQ ID NO:7, had positive effects on properties such asexpression, stability, and viscosity of a bispecific molecule. Thus, avariety of proteins containing these linkers may have such favorableproperties as compared to similar proteins containing other linkers.

Therapeutic Uses of Bispecific Proteins

The bispecific proteins binding to BAFF and B7RP1 described herein canbe used as therapeutics for a variety of indications, particularlyconditions driven by auto-antibodies and/or conditions mediated by bothT cells and B cells. Such conditions include, for example, SLE, lupus,nephritis, ANCA-positive vasculitis, rheumatoid arthritis (RA),dermatomyositis, polymyositis, gastrointestinal diseases such as Crohn'sdisease, ulcerative colitis, and celiac disease, skin conditions such aspemphigus, pemphigoid, and subacute cutaneous lupus erythematosus(SCLE), diseases of the nervous system such as multiple sclerosis andchronic inflammatory demyelinating polyneuropathy (CIDP), neuromusculardiseases such as myasthenia gravis, diseases involving the kidneys suchas Goodpasture's syndrome and glomerulonephritis, hematologic conditionssuch as autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenicpurpura (ITP), and autoimmune neutropenia, liver conditions such aschronic active hepatitis and primary biliary cirrhosis, Sjogren'ssyndrome, systemic sclerosis, and endocrine conditions includingHashimoto's thyroiditis, Graves' disease, Addison's disease, andmultiple endocrine autoimmune failure (commonly including diabetes,hypothyroidism, Addison's disease, and gonadal failure). Atherapeutically effective amount of a bispecific protein as describedherein can be administered to a patient suffering from any of theseconditions to treat the condition.

In one embodiment, a bispecific protein that can inhibit BAFF-mediated Bcell proliferation and B7RP1-mediated T cell proliferation can be usedto treat a patient suffering from SLE. SLE is an autoimmune disease ofunknown etiology marked by autoreactivity to nuclear self antigens. Itsclinical manifestations are so diverse that it is questionable whetherit is truly a single disease or a group of related conditions. Kotzin(1996) Systemic lupus erythematosus. Cell 85: 303-306; Rahman andIsenberg (2008), Systemic lupus erythematosus. N. Engl J. Med. 358:929-939. Symptoms can include the following: constitutional symptomssuch as malaise, fatigue, fevers, anorexia, and weight loss; diverseskin symptoms including acute, transient facial rashes in adults,bullous disease, and chronic and disfiguring rashes of the head andneck; arthritis; muscle pain and/or weakness; cardiovascular symptomssuch as mitral valve thickening, vegetations, regurgitation, stenosis,pericarditis, and ischemic heart disease, some of which can culminate instroke, embolic disease, heart failure, infectious endocarditis, orvalve failure; nephritis, which is a major cause of morbidity in SLE;neurological symptoms including cognitive dysfunction, depression,psychosis, coma, seizure disorders, migraine, and other headachesyndromes, aseptic meningitis, chorea, stroke, and cranial neuropathies;hemotologic symptoms including leucopenia, thrombocytopenia, serositis,anemia, coagulation abnormalities, splenomegaly, and lymphadenopathy;and various gastrointestinal abnormalities. Id; Vratsanos et al.,“Systemic Lupus Erythematosus,” Chapter 39 in Samter's ImmunologicalDiseases, 6^(th) Edition, Austen et al., eds., Lippincott Williams &Wilkins, Phiiladelphia, P A, 2001. Severity of symptoms varies widely,as does the course of the disease. SLE can be deadly.

An SLE patient can be treated with a bispecific protein that inhibitsBAFF and B7RP1 before, after, or concurrently with treatment using anexisting therapy for SLE. Such existing therapies for SLE includecorticosteroids such as prednisone, prednisolone, andmethylprednisolone, antimalarials such as hydroxychloroquine,quinacrine, and chloroquine, retinoic acid, aspirin and othernonsteroidal anti-inflammatory drugs (NSAIDs), cyclophosphamide,dehydroepiandrosterone, mycophenolate mofetil, azathioprine,chlorambucil, methotrexate, tacrolimus, dapsone, thalidomide,leflunomide, cyclosporine, belimumab, anti-CD20 antibodies such asrituximab, and fusion proteins such as abatacept.

The disease activity of SLE patients can be rated using an instrumentsuch as the Systemic Lupus Erythrmatosus Disease Activity Index(SLEDAI), which provides a score for disease activity that takes intoconsideration the following symptoms, which are weighted according toseverity: seizure, psychosis, organic brain syndrome, visualdisturbance, cranial nerve disorder, lupus headache, vasculitis,arthritis, myositis, urinary casts, hematuria, proteinuria, pyuria, newrash, alopecia, mucosal ulcers, pleurisy, pericarditis, low complement,increased DNA binding, fever, thrombocytopenia, and leucopenia.Bombardier et al. (1992), Arthr. & Rheum. 35(6): 630-640, the relevantportions of which are incorporated herein by reference. The treatmentsdescribed herein can be useful in lessening or eliminating symptoms ofSLE as measured by SLEDAI. Methods of treatment described herein canimprove a patient's SLEDAI score compared to a baseline value for thesame patient prior to initiation of treatment with a bispecific proteinas described herein.

Another method for assessing disease activity in SLE is the BritishIsles Lupus Assessment Group (BILAG) index, which is a disease activityassessment system for SLE patients based on the principle of thephysician's intention to treat. Stoll et al. (1996), Ann. Rheum Dis. 55:756-760; Hay et al. (1993), Q. J. Med. 86: 447-458. The portions ofthese references describing the BILAG are incorporated herein byreference. A BILAG score is assigned by giving separate numeric oralphabetic disease activity scores in each of eight organ-based systems,general (such as fever and fatigue), mucocutaneous (such as rash andalopecia, among many other symptoms), neurological (such as seizures,migraine headaches, and psychosis, among many other symptoms),musculoskeletal (such as arthritis), cardiorespiratory (such as cardiacfailure and decreased pulmonary function), vasculitis and thrombosis,renal (such as nephritis), and hematological. Id. The treatmentsdescribed herein can be useful in lessening or eliminating symptoms ofSLE as measured by the BILAG index or in decreasing a patient's BILAGscore as compared to a baseline value prior to the initiation oftreatment with a bispecific protein as described herein.

A bispecific protein as described herein, which inhibits BAFF-mediatedproliferation of B cells and B7RP1-mediated proliferation of T cells,could also be used to treat rheumatoid arthritis (RA). RA is a chronicdisease with systemic symptoms, as well as symptoms relatingspecifically to the joints. Symptoms commonly include synovitis, leadingto painful and swollen joints, and various laboratory abnormalities suchas higher-than-normal levels of rheumatoid factor, anti-citrullinemodified protein (anti-CCP) antibodies, and C-reactive protein (CRP) andan elevated erythrocyte sedimentation rate (ESR). Less common symptomsinclude various extra-articular symptoms involving, e.g., tendons,ligaments, blood vessels, the heart, and the lungs. Disease activity canbe often measured using a variety of indices. See, e.g., Anderson et al.(2012), Arthritis care & Res. 64 (5): 640-647, the portions of whichdiscuss such indices are incorporated herein by reference. Elementsincluded in such scoring indices include the number of tender joints,the number of swollen joints, functional assessments, and variouslaboratory findings such as CRP, ESR, etc.

In some embodiments, a patient suffering from RA can be treated with abispecific protein that inhibits BAFF-mediated B cell proliferation andB7RP1-mediated T cell proliferation before, after, or concurrently withtreatment with a drug in current use for RA. Therapeutics currently inuse for rheumatoid arthritis (RA) include non-steroidalanti-inflammatory drugs (NSAIDs) (such aspirin and cyclooxygenase-2(COX-2) inhibitors), disease modifying anti-inflammatory drugs (DMARDs,such as methotrexate, leflunomide, and sulfasalazine), anti-malarials(such as hydroxychloroquine), cyclophosphamide, D-penicillamine,azathioprine, gold salts, tumor necrosis factor inhibitors (such asetanercept, infliximab, adalimumab, golimumab, and certolizumab pegol),CD20 inhibitors such as rituximab, IL-1 antagonists such as anakinra,IL-6 inhibitors such as tocilizumab, inhibitors of Janus kinases (JAKs,such as tofacitinib), abatacept, and corticosteroids, among others.

A therapeutically effective amount of a bispecific protein as describedherein, which inhibits BAFF-mediated proliferation of B cells andB7RP1-mediated proliferation of T cells, can also be used to treat aninflammatory bowel disease, such as Crohn's disease or ulcerativecolitis. Crohn's disease involves an abnormal inflammation of anyportion of the alimentary tract from the mouth to the anus, although inmost patients abnormal inflammation is confined to the ileocolic,small-intestinal, and colonic-anorectal regions. Typically, theinflammation is discontinuous. Common symptoms include abdominal pain,anorexia, weight loss, fever, diarrhea, fullness and/or tenderness inthe right lower quadrant of the abdomen, constipation, vomiting, andperianal discomfort and discharge. Other possible symptoms includeperipheral arthritis, growth retardation, episcleritis, aphthousstomatitis, erythema nodosum, pyoderma gangrenosum, kidney stones,impaired urinary dilution and alkalinization, malabsorption, andgallstones, among others. See e.g. Strober et al., Medical Immunology,10^(th) Edition, Section III, Ch. 35 (2001); Merck Manual of Diagnosisand Therapy, 17^(th) Edition, Section 3, Ch. 31 (1999). Macrophagesisolated from patients with Crohn's disease produce increased amounts ofIL-12, IFNγ, INFα, and other inflammatory cytokines.

Ulcerative colitis, though it is sometimes hard to distinguish fromCrohn's disease, is distinct from Crohn's disease in several respects.First, it is generally limited to the colon while Crohn's disease mayoccur throughout the alimentary tract. Second, ulcerative colitis mainlyinvolves inflammation only of the superficial layers of the bowel,unlike Crohn's disease in which the inflammation can penetrate all waythrough the wall of the bowel or other location in the alimentary tract.Finally, ulcerative colitis typically involves a continuous area ofinflammation, rather than the discontinuous sites of inflammationtypical of Crohn's disease. Like Crohn's disease, ulcerative colitis isfound primarily in urban areas. Also, genetic factors likely play a rolein ulcerative colitis since there is a familial aggregation of cases.Autoantibodies are observed in ulcerative colitis patients more oftenthan Crohn's disease patients. The autoantibodies are often directed tocolonic epithelial cell components. Among the most common areantineutrophil cytoplasmic antibodies with specificities for catalase,α-enolase, and lactoferrin. In some cases such antibodies cross reactwith colonic microorganisms.

In clinical trials, Crohn's disease activity is often scored using theCrohn's Disease Activity Index (CDAI). The CDAI provides a diseaseactivity score based on eight factors including (1) the number of liquidor soft stools per day, (2) a patient rating of the amount of abdominalpain per day, (3) a patient rating of general well-being, (4) a patientreport of other symptoms including arthritis, iritis, uveitis, erythemanodosum, pyoderma gangrenosum, ephthous stomatitis, anal fissure,fitula, or abscess, other fistula, or fever, (5) patient report oftaking lomotil or other opiates for diarrhea, (6) abdominal mass, (7)hematocrit, and (8) body weight. See, e.g., Best et al. (1976),Gastroenterol. 70: 439-444, the relevant portions of which areincorporated herein by reference.

Symptoms of ulcerative colitis are variable. They may include diarrhea,tenesmus, abdominal cramps, blood and mucus in the stool, fever, andrectal bleeding. Toxic megacolon, a potentially life-threateningcondition in which the colon is dilated beyond about 6 centimeters andmay lose its muscular tone and/or perforate, may also occur. Othersymptoms that may accompany ulcerative colitis include peripheralarthritis, ankylosing spondylitis, sacroiliitis, anterior uveitis,erythema nodosum, pyoderma gangrenosum, episcleritis, autoimmunehepatitis, primary sclerosing cholangitis, cirrhosis, and retardedgrowth and development in children.

In some embodiments a patient suffering from an inflammatory boweldisease (IBD), such as Crohn's disease or ulcerative colitis, can betreated with a bispecific protein that binds to BAFF and B7RP1 before,after, or concurrently with treatment with an existing therapy for IBD.Existing therapeutics for IBD include, for example, sulfasalazine,5-aminosalicylic acid and its derivatives (such as olsalazine,balsalazide, and mesalamine), anti-TNF antibodies (including infliximab,adalimumab, golimumab, and certolizumab pegol), corticosteroids for oralor parenteral administration (including prednisone, methylprednisone,budesonide, or hydrocortisone), adrenocorticotropic hormone, antibiotics(including metronidazole, ciprofloxacin, or rifaximin), azathioprine,6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, andthalidomide.

Nucleic Acids Encoding Bispecific Proteins

Provided herein are nucleic acids encoding a bispecific protein that caninhibit B7RP1-mediated T cell proliferation and BAFF-mediated B cellproliferation. For example, SEQ ID NO:52 encodes the VL region havingthe amino acid sequence of SEQ ID NO:14, and SEQ ID NO:53 encodes the VHregion having the amino acid sequence of SEQ ID NO:15. Similarly, SEQ IDNOs:55 and 56 encode the amino acid sequences of SEQ ID NOs:17 and 18,respectively, which are polypeptides comprising the heavy chain of ananti-B7RP1 antibody fused to two BAFF-binding peptides. SEQ ID NO:57encodes the light chain of an anti-B7RP1 antibody, which can be part ofa hetero-tetrameric bispecific IgG antibody or a bispecific fusionprotein, as described above. Any nucleic acid sequence encoding anyamino acid sequence provided herein is contemplated. Similarly,nucleotide sequence variants including silent mutations relative tosequences disclosed herein or encoding the amino acid sequence variantsdescribed above are also included within the ambit of the invention.More specifically, nucleotide sequences encoding amino acid sequencesthat vary by no more than 10 insertions, deletions, or substitutions ofa single amino acid per 100 amino acids from amino acid sequencesdisclosed herein are contemplated.

Nucleic acid sequences encoding bispecific proteins described herein canbe determined by one of skill in the art based on the amino acidsequences provided herein and knowledge in the art. Besides moretraditional methods of producing cloned DNA segments encoding aparticular amino acid sequence, companies such as DNA 2.0 (Menlo Park,Calif., USA) and BlueHeron (Bothell, Wash., USA), among others, nowroutinely produce chemically synthesized, gene-sized DNAs of any desiredsequence to order, thus streamlining the process of producing such DNAs.Codon usage can be adjusted so as to optimize expression in the systemof choice.

Methods of Making Bispecific Proteins that Bind to BAFF and B7RP1

Nucleic acids encoding the bispecific proteins described herein can beinserted into vectors appropriate for the host cell in which the nucleicacid will be expressed. These nucleic acids can be introduced into thehost cells by any of the methods well-known in the art. Host cells thatcan be used include bacteria, including Escherichia coli, yeast,including Saccharomyces cerevisiae or Pichia pastoris, insect cellsincluding Spodoptera frugiperda cells, plant cells, and mammalian cells,including Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK)cells, monkey kidney cells, HeLa cells, human hepatocellular carcinomacells, and 293 cells, among many others. These host cells can becultured under conditions such that the introduced nucleic acids will beexpressed, and the bispecific protein can be recovered from the culturesupernatant or the cell mass.

Generally, the procedure used to introduce the nucleic acids into thehost cells may depend upon the host cell into which the nucleic acidsare to be introduced. Methods of introducing nucleic acids into bacteriaare well-known in the art. For example, electroporation or calciumchoride transformation are commonly used. Methods for introduction ofnucleic acids into yeast are also well-known in the art and include, forexample, transformation methods using lithium acetate and polyethyleneglycol. Methods for introducing heterologous polynucleotides intomammalian cells are well known in the art and include, but are notlimited to, dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Expression vectors used in any of the host cells can contain sequencesnecessary for DNA replication, selection of host cells containing thevector, and expression of the exogenous nucleotide sequences. Suchsequences can typically include one or more of the following nucleotidesequences: a promoter, one or more enhancer sequences, an origin ofreplication, a transcriptional termination sequence, a complete intronsequence containing a donor and acceptor splice site, a sequenceencoding a leader sequence for polypeptide secretion, a ribosome bindingsite, a polyadenylation sequence, a polylinker region for inserting thenucleic acid encoding the polypeptide to be expressed, and a selectablemarker element. Numerous expression vectors appropriate for expressionin various host cells are known in the art and are commerciallyavailable.

Pharmaceutical Compositions, Dosing, and Methods of Administration

Pharmaceutical compositions comprising the bispecific proteins describedherein are provided. Such compositions can comprise a therapeuticallyeffective amount of a bispecific protein with one or more additionalcomponents such as a physiologically acceptable carrier, excipient, ordiluent. Such additional components can include buffers, carbohydrates,polyols, amino acids, chelating agents, stabilizers, and/orpreservatives, among many possibilities. Many such additional componentsare described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th)Edition, (A. R. Gennaro, ed.), 1990, Mack Publishing Company, therelevant portions of which are incorporated herein by reference.

Dosing of the bispecific proteins described herein can be adjusted toachieve the desired effects. In many cases, repeated dosing will berequired because of the chronic nature of the disease being treated. Forexample, a bispecific protein as described herein can be dosed twice perweek, once per week, once every two, three, four, five, six, seven,eight, nine, or ten weeks, or once every two, three, four, five, or sixmonths. The amount of the bispecific protein administered on each daythat it is administered can be from about 0.0036 mg to about 700 mg.Alternatively, the dose can calibrated according to the estimated skinsurface of a patient, and each dose can be from about 0.002 μg/m² toabout 350 mg/m². In another alternative, the dose can be calibratedaccording to a patient's weight, and each dose can be from about0.000051 mg/kg to about 10.0 mg/kg.

The bispecific proteins, or pharmaceutical compositions containing thesemolecules, can be administered by any feasible method. Therapeutics thatcomprise a protein will ordinarily be administered by a parenteralroute, for example by injection, since oral administration, in theabsence of some special formulation or circumstance, would lead tohydrolysis of the protein in the acid environment of the stomach.Subcutaneous, intramuscular, intravenous, intraarterial, intralesional,and peritoneal bolus injections are possible routes of administration.The bispecific proteins can also be administered via infusion, forexample intravenous or subcutaneous infusion. Topical administration isalso possible, especially for diseases involving the skin.Alternatively, the bispecific proteins can be administered throughcontact with a mucus membrane, for example by intra-nasal, sublingual,vaginal, or rectal administration or administration as an inhalant.Alternatively, certain appropriate pharmaceutical compositionscomprising a bispecific protein can be administered orally.

Having described the invention in general terms above, the followingexamples are offered by way of illustration and not limitation.

EXAMPLES Example 1: Designing and Testing a BAFF/B7RP1 BispecificMolecule for Human Therapeutic Use

The object of this series of experiments was to find a bispecificmolecule that (1) inhibits BAFF-mediated B cell proliferation andB7RP1-mediated T cell proliferation, (2) is highly active in biologicalassays, and (3) has favorable biophysical properties. A number ofschematic designs for the fusion of a peptide that binds human BAFF toan anti-human B7RP1 IgG antibody (anti-huB7RP1) are illustrated inFIG. 1. The sequence of the BAFF-binding peptide is provided in SEQ IDNO:1, and the sequences of the immunoglobulin heavy and light chains ofanti-huB7RP1 are provided in SEQ ID NO:25 and SEQ ID NO:19,respectively.

To determine which design had the best biophysical properties, whileretaining biological activity, the bispecific molecules diagrammed inFIG. 1 were made and tested. In one construct, two tandem copies of theBAFF-binding peptide with an intervening linker (the “1K linker,” havingthe amino acid sequence of SEQ ID NO:24) were fused to the N-terminus ofeither the immunoglobulin heavy chain (P71617) or immunoglobulin lightchain (P71618) of anti-huB7RP1. See FIG. 1. The amino acid sequence ofthe P71617 heavy chain is provided in SEQ ID NO:26, and the amino acidsequence of the light chain of P71617 is the same as that of theimmunoglobulin light chain of anti-huB7RP1 (SEQ ID NO:19). The aminoacid sequence of the P71618 light chain is provided in SEQ ID NO:27, andthe amino acid sequence of the heavy chain of P71618 is the same as theimmunoglobulin heavy chain of anti-huB7RP1 (SEQ ID NO:25). Two tandemcopies of the BAFF-binding peptide were also fused to the C-terminal endof the immunoglobulin heavy chain of anti-huB7RP1 (having the amino acidsequence of SEQ ID NO:25) using either the 1K linker mentioned above(having the amino acid sequence of SEQ ID NO:24; P71619) or a 5X(G4S)linker (SEQ ID NO: 71) between the two BAFF-binding peptides (P71620).The amino acid sequences of the heavy chains of these two fusionconstructs are provided in SEQ ID NO:16 (P71619) and SEQ ID NO:28(P71620). In construct P71621, two tandem copies of the BAFF-bindingpeptide with an intervening 1K linker were inserted into the antibody'sCH3 domain between residues 358 and 359 of the amino acid sequence ofSEQ ID NO:25 (the amino acid sequence of the immunoglobulin heavy chainof the anti-huB7RP1 antibody). The sequence of the heavy chain of theP71621 construct is provided in SEQ ID NO:29. In construct P71622, theBAFF-binding peptide was inserted into the CH3 domain of theimmunoglobulin heavy chain of anti-huB7RP1 (between residues 358 and 359of SEQ ID NO:25 and a second copy of the BAFF-binding peptide was fusedto the C-terminal end of the heavy chain. The amino acid sequence of theheavy chain of P71622 is provided in SEQ ID NO:30. In construct P71623,one BAFF-binding peptide was inserted into the CH2 region (betweenresidues 268 and 269 of SEQ ID NO:25), and a second BAFF-binding peptidewas inserted into the CH3 region (between residues 358 and 359 of SEQ IDNO:25). SEQ ID NO:31 is the amino acid sequence of the heavy chain ofP71623. Constructs P71619-P71623 all have the immunoglobulin light chainof anti-huB7RP1 (SEQ ID NO:19).

In constructs P74293 and P74294, the linker between the two tandemcopies of the BAFF-binding peptides in construct P71619 was modified.The amino acid sequences of the heavy chains of P74293 and P74294 areprovided in SEQ ID NO:17 and SEQ ID NO:18, respectively. Theimmunoglobulin light chains of these constructs also have the amino acidsequence of SEQ ID NO:19.

Nucleic acids encoding the constructs described above were made asfollows. Nucleic acids encoding the N-terminal portion of the N-terminalBAFF peptide fusions (P71617 and P71618), including two copies of theBAFF-binding peptide plus an immunoglobulin heavy or light chainvariable region, were generated synthetically. These were ligated,through convenient restriction endonuclease sites, to nucleic acidsencoding the immunoglobulin heavy or light chain constant region inappropriate vectors. Nucleic acids encoding the heavy chain constantregion C-terminal fusions (P71619 and P71620), Fc-loop insertions(P71621 and P71623), and the Fc-loop insertion/C-terminal fusion(P71622) were all generated synthetically and ligated into a vectorcontaining the heavy chain variable region through convenientrestriction endonuclease sites.

The various bispecific constructs described above were expressed in bothtransiently transfected 293 cells and stably transfected CHO cells. Thefusion proteins were purified and tested for biological activity. Nodifferences were observed in proteins produced in these two differentkinds host cells.

The BAFF inhibitory activities of the bispecific molecules were testedin a BAFF-mediated human primary B cell proliferation assay. In brief,human B cells were purified from peripheral blood mononuclear cells(PBMCs) using negative selection using a human B cell kit II fromMiltenyi Biotec (Auburn, Calif.). About 10⁵ purified B cells werecultured in 96 well microtiter plates in Minimal Essential Media (MEM)plus 10% heat inactivated fetal bovine serum (FBS) in the presence of 50ng/ml human BAFF protein, 2 μg/ml goat F(ab′) 2 anti-human IgM (JacksonImmunoResearch), and varying concentrations of one of the bispecificproteins described above at 37° C. in 5% CO² for 48 hours. An anti-BAFFpeptibody was used as a positive control (“αBAFF,” which is a homodimercontaining two polypeptide chains, each comprising two BAFF-bindingpeptides fused to an Fc polypeptide). The αBAFF molecule is described indetail in U.S. Pat. No. 7,259,137, and the amino acid sequence of onepolypeptide chain of this homodimer is provided in SEQ ID NO:32. Theportions of U.S. Pat. No. 7,259,137 describing αBAFF are incorporatedherein by reference. Proliferation was measured by the uptake ofradioactive ³H-thymidine during the last 18 hours of incubation. Resultsare shown in FIGS. 2A and 2B.

The data in FIG. 2A indicate that the two C-terminal fusion constructs(P71619 and P71620) were comparable to each other in inhibition ofBAFF-mediated B cell proliferation and more potent than all of the otherfusion constructs tested in this experiment. P71620 was not pursuedfurther because it tended to aggregate, a property that is highlyundesirable in a therapeutic protein. The data in FIG. 2B indicate thatP71619 is comparable to the two slightly modified versions of thisconstruct described above (P74293 and P74294) and to a positive control(αBAFF) in inhibition of BAFF-mediated B cell proliferation. Thus, amongthe bispecific constructs tested, P71619, P71620, P74293, and P74294 hadcomparable activity in this assay of BAFF-mediated B cell proliferationand better activity than all other constructs tested.

The B7RP1 inhibitory activity of P71619, P74293, and P74294 was assayedusing a human B7RP1-Fc-mediated T cell proliferation assay. Primaryhuman T cells purified from PBMCs from healthy human donors using Pan Tcell isolation kit from Miltenyi Biotec (Auburn, Calif.) and stimulatedwith plate-bound anti-CD3 (1 μg/mL) antibody and a B7RP1-Fc fusionprotein (3 μg/mL) in the presence of varying concentrations of thebispecific proteins described above or an IgG2 anti-human B7RP1 antibody(referred to herein as “αB7RP1”). ³H-thymidine was added to the cellsafter 48 hours, and incorporation of the ³H-thymidine was measured 24hours later. All of the bispecific antibodies that were tested hadsimilar IC₅₀'s, which were similar to that of αB7RP1 (FIG. 3). Thus,these data suggest that the conjugation of the BAFF-binding peptides tothe anti-huB7RP1 antibody had little or no effect on the ability of theantibody to inhibit B7RP1 activity.

The binding affinities of the heterodimeric bispecific antibodies P74293and P74294 to BAFF and B7RP1 were measured by Kinetic Exclusion Assay(KinExA®; Sapidyne Instruments, Boise, Id.). Both antibodies have highbinding affinities to human BAFF (having K_(d)'s of approximately 30 pM)and to human B7RP1 (having K_(d)'s of approximately 40 pM). See Table 2below. In addition, both of these bispecifics have similar bindingaffinities to cynomolgus monkey BAFF compared to human BAFF and tocynomolgus monkey B7RP1 compared to human B7RP1. Table 2.

TABLE 2 binding affinity and cellular potency of P74293 and P74294.P74293 P74294 K_(d) (pM) for binding to human BAFF 29 37 K_(d) (pM) forbinding to cynomolgus monkey 22.3 17.4 BAFF IC₅₀ (nM) for inhibition ofBAFF-mediated 0.86 0.96 human B cell proliferation IC₅₀ (nM) forinhibition of BAFF-mediated 1.6 1.8 cynomolgus monkey B cellproliferation K_(d) (pM) for binding to human B7RP1 38 41 K_(d) (pM) forbinding to cynomolgus monkey 49.4 45.2 B7RP1 IC₅₀ (nM) for inhibition ofB7RP1-mediated 1.36 0.98 human T cell proliferation IC₅₀ (nM) forinhibition of B7RP1-mediated 0.29 ND* cynomolgus monkey T cellproliferation *ND means not determined.

To further assess the activity of P74293 in an in vitro system usinghuman cells, cytokine production by human tonsil cells activated byStaphylococcus enterotoxin B (SEB) was assessed in the presence ofvarious test molecules. Briefly, human tonsil cells were isolated fromtissue and stimulated with SEB (1 μg/mL) in the presence of one of thefollowing molecules: (1) αB7RP1, (2) P74293, (3) CTLA4-Ig (a positivecontrol), or (4) human IgG (a negative control). After 72 hours ofculture, the cell supernatant was collected, and cytokine levels wereassayed using kits from Meso Scale Discovery according to themanufacturer's instructions. Results are shown in FIG. 4.

All three of αB7RP1, P74293, and CTLA4-Ig, bars 1, 2, and 3,respectively in all panels of FIG. 4, inhibited release of IL-17, IL-10,IL-4, and IFNγ. Release of IL-2 was inhibited only by CTLA4-Ig. Thus,αB7RP1 and the anti-BAFF/B7RP1 bispecific P74293 had comparable andspecific effects on cytokine secretion by SEB-activated human tonsilcells.

Three heterodimeric bispecific proteins, that is, P71619, P74293, andP74294, were examined for additional properties. Protein titers fromcultures of host cells producing these proteins indicated that P74293and P74294 were produced at about twice the levels at which P71619 wasproduced. P74293 and P74294 were also more stable than P71619 afterstorage for two weeks at 40° C. as assessed by size exclusionchromatography (SEC). P74293 formed a clear solution at the onset ofstorage and after 4 weeks of storage, whereas solutions containingP74394 were hazy at all time points. Solutions of P74293 and P74294 wereless viscous than solutions of P71619. Thus, P74293 and P74294 wereexpressed at higher levels than P71619 and were also more stable andless viscous in the concentration range tested than P71619. The mostobvious difference between these molecules lies in the linker betweenthe two BAFF-binding peptides. These data suggest that the linkers inP74293 and P74294 (SEQ ID NOs:6 and 7) can confer improved propertiesupon these molecules.

The pharmacokinetic properties of the bispecific molecules describedwere evaluated in mice. Male CD-1 mice were given a single intravenous(IV) dose (5 mg/kg) of the bispecific fusion proteins P71617, P71619,P71621, P71622, P74293, or P74294. Serum samples were collected beforedosing and at 0.5, 2, 8, 24, 48, 72, 96, 168, 240, 336, 408, 504 hoursafter dosing. The concentration of the bispecific molecule in the serumwas determined by two ELISA methods, one registering the presence of theFc portion and one registering the presence of both the Fc portion andthe BAFF-binding peptide portion. For the Fc portion measurement, abiotinylated anti-Fc antibody was used as capture reagent, and ALEXAFLUOR® 647-labeled anti-Fc antibody was used as the detection reagent.To detect the BAFF-binding portion and the Fc portion of the bispecific,a biotinylated BAFF protein was used as the capture reagent, and ALEXAFLUOR® 647-labeled anti-Fc antibody was used as the detection reagent.The bispecific proteins with two tandem copies of BAFF-binding petidesfused to the N-terminus (P71617), C-terminus (P71619, P74293 and P74294)or CH3 domain (P71621) of the heavy chain have very similar PK profilesin mice. FIG. 5. The bispecific protein with one copy of BAFF-bindingpeptide inserted into the CH3 domain and another copy fused to theC-terminal end of the heavy chain (P71622) had lower exposure comparedto the other bispecific proteins. FIG. 5. In addition, the two differentELISA assays resulted in similar serum concentrations of the bispecificproteins, suggesting that no significant cleavage of the bispecificproteins occurred in vivo.

Pharmacokinetic and pharmacodynamic parameters of the P74293 and P74294heterodimeric bispecific antibodies were also assessed by a single dosestudy in cynomolgus monkeys. Naïve male cynomolgus monkeys (n=4) weregiven a single bolus intravenous or subcutaneous dose of P74293 (10mg/kg), or a single subcutaneous dose of P74294 (10 mg/kg). Bothbispecific molecules have PK profiles similar to that of an IgGantibody. The observed pharmacokinetic parameters for P74293 and P74294,as well as for anti-huB7RP1, are reported in Table 3 below.

TABLE 3 Pharmacokinetic parameters in cynomolgus monkey P74293 P74294Anti-huB7RP1 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg IV SC SC IV SCMaximum drug 323 90 74 264 112 concentration (C_(max); μg/ml) Time atwhich C_(max) was 45 51 72 observed (T_(max); hr) Area under the curve33800 20300 22000 26100 23800 (AUC_(0-inf); μg*hr/mL) Mean residencetime 136 132 148 138 144 (MRT_(0-inf); hr) Total clearance (CL; 0.3030.491 0.484 0.388 0.427 ml/hr/kg) Volume of distribution at 42.5 52.1steady state (Vss; ml/kg)The data in Table 3 indicate that the pharmacokinetic parameters ofP74293 and P75294 are comparable to each other and to those ofanti-huB7RP1 antibody.

Example 2: Designing and Testing a Murine Bispecific Surrogate Molecule

To conduct preclinical studies in mice, a murine surrogate bispecificmolecule that could bind to murine B7RP1 and murine BAFF (hereinafter,the “murine surrogate”) was constructed. The anti-huB7RP1 antibody usedto construct the bispecific constructs described in Example 1, does notbind to murine B7RP1, while the BAFF-binding peptide used in theseconstructs does bind to both human and murine BAFF. Data not shown. Themurine surrogate comprises an antagonistic IgG anti-murine B7RP1antibody (called “anti-mB7RP1” herein), which was a chimera of mouseimmunoglobulin constant regions and rat anti-murine B7RP1 immunoglobulinvariable regions. The use of anti-mB7RP1 is described in Hu et al.(2009), J. Immunol. 182: 1421, where it is designated 1B7-V2. The murinesurrogate has two copies of a BAFF-binding peptide (SEQ ID NO:1) fusedvia a short linker (five amino acids long) to the C-terminus of theimmunoglobulin heavy chain of anti-mB7RP1. The two copies of theBAFF-binding peptide are separated by another linker that is 23 aminoacids long. Nucleic acids encoding the heavy chain of the murinesurrogate were made using overlap PCR to join nucleic acids encoding theBAFF-binding portion of αBAFF to the downstream end of nucleic acidsencoding the heavy chain of 1B7-V2, i.e., anti-mB7RP1.

BAFF inhibition by the murine surrogate was evaluated in a BAFF-mediatedB cell proliferation assay. Mouse B lymphocytes were isolated fromC57BL/6 spleens by negative selection with MACS CD43 (ly-48) Microbeadsaccording to the manufacturers instructions (Miltenyi Biotec, Auburn,Calif.) or from PBMC using a B cell isolation kit (Miltenyi Biotec,Auburn, Calif.). Purified B cells were stimulated with 0.1 μg/mlanti-IgM and 200 ng/ml BAFF in the presence of varying concentrations ofthe murine surrogate or αBAFF. B cell proliferation was measured by³H-thymidine incorporation at day 4. The IC₅₀'s of the murine surrogateand αBAFF were 0.59 nM and 0.73 nM, respectively. See FIG. 6A. Thus, themurine surrogate effectively inhibited BAFF with potency comparable tothat of αBAFF.

To measure inhibition of B7RP1 binding to its receptor by the murinesurrogate, mouse spleen cells were first activated to enhance theirexpression of the B7RP1 receptor by incubating them in microtiter wellscoated with an anti-CD3 (5 μg/ml) antibody for 24 hours. The activatedspleen cells were washed with phosphate buffered saline (PBS) and thenincubated with 5 μg/ml biotinylated muB7RP1:Fc in the presence ofvarying concentrations of the murine surrogate at 4° C. for 30 minutes.The cells were washed and then stained with allophycocyanin(APC)-conjugated anti-mouse CD3 antibody and streptavidin-phycoerythrin(Streptavidin-PE) for an additional 20 minutes. The B7RP1-Fc binding toT cells was analyzed by flow cytometry. The IC₅₀'s of the murinesurrogate and anti-mB7RP1 were 4.01 pM and 2.8 pM, respectively. SeeFIG. 6B. Hence, the activity of the murine surrogate was similar to thatof anti-mB7RP1 in this assay. Thus, the murine surrogate inhibits bothBAFF and B7RP1.

The in vivo pharmacodynamic effects of the murine surrogate wereevaluated in mice immunized with the sheep red blood cells (SRBC). Inbrief, BALB/c mice (8 weeks old) received a primary immunization on day0 and a booster immunization on day 28 with 2×10⁸ SRBC in 0.2 ml of PBSvia intraperitoneal injection. The mice (n=5 for each molecule) weretreated twice per week from day 0 to day 33 with one of the followingmolecules at 5 mg/kg: the murine surrogate; αBAFF; anti-mB7RP1; ormurine IgG1. Mice treated with SBRC, but not receiving anothertreatment, served as positive controls. The mice were sacrificed on day34, and serum and spleens were collected.

To measure the proportion of B cells and memory T cells in the spleen,spleen cells were harvested by grinding the spleen tissue through a cellstrainer. The spleen cells were preincubated with unlabelledanti-CD16/32 to block the nonspecific binding of antibodies to Fc gammareceptors (FcγR). The proportion of B cells was determined by stainingwith PE-labeled anti-B220 (which is expressed on B cells). Theproportion of memory T cells cells (CD44^(hi)CD62L^(lo)CD4 T cells) wasdetermined by staining with FITC-conjugated anti-CD44, PE-conjugatedanti-CD62L, APC-conjugated anti-CD4 and PerCP-conjugated anti-CD3. Allstaining antibodies were purchased from BD Bioscience (San Diego,Calif.). For both B and T cell determinations, flow cytometry wasperformed with a FACSCALIBUR™ (BD Bioscience, San Jose, Calif.) flowcytometer, and the data was analyzed using FLOWJO® (TreeStar Inc.,Ashland, Oreg.) software for analysis of flow cytometry data. Resultsare shown in FIG. 7.

To measure levels of anti-SBRC antibodies in serum, microtiter platescoated with 10 μg/ml soluble SRBC antigen were incubated for two hoursat room temperature with diluted serum from treated mice. BoundSRBC-specific Ig from the serum was detected with HRP-conjugatedpolyclonal goat anti-mouse IgG and IgM antibodies (Southern Biotech,Birmingham, Ala.). The substrate reaction was performed using SUREBLUE™TMB microwell peroxidase substrate (KPL, Gaithersburg, Md.) according tothe manufacturer's instructions, and the optical density was read usinga Spectrum Max microplate reader (Molecular Devices). As a positivecontrol, serial dilutions of a mixture of sera from SRBC-immunized micewithout any treatment was added to each plate, and a standard curve wasconstructed from the readings from these wells. Levels of anti-SBRCantibodies of other samples are reported in FIG. 7 as a percentage ofthis positive control.

The percentage of spleen cells that are B cells was reduced in micetreated with the murine surrogate as compared to the percentage observedin mice treated with murine IgG1. FIG. 7 (top panel). A similarreduction was observed in mice treated with αBAFF or αBAFF plusanti-mB7RP1, but not in mice treated with anti-mB7RP1 alone. FIG. 7 (toppanel). With regard to memory T cells, mice treated with the murinesurrogate, anti-mB7RP1, or anti-mB7RP1 plus αBAFF had reducedproportions of memory T cells compared to that observed in mice treatedwith muIgG1. FIG. 7 (middle panel). In contrast, treatment with αBAFFdid not alter the memory T cell population in spleen compared to thatobserved with muIgG treatment. FIG. 7 (middle panel). The murinesurrogate also showed potent reduction of the anti-SRBC antibody levelin serum, similar to that observed upon treatment with anti-mB7RP1 oranti-mB7RP1 plus αBAFF or in mice that had not been injected with SRBC.FIG. 7 (bottom panel). Moderate inhibition of anti-SRBC antibody level,compared to the level observed with mIgG1 treatment, was observed inmice treated with αBAFF alone. FIG. 7 (bottom panel). These dataindicate that the murine surrogate had dual inhibitory effects in B celland T cell compartments in mice in vivo.

The impact of the murine surrogate on disease was evaluated in the NZB/WF₁ lupus model using two different dose amounts for each of themolecules tested. Female NZB/W F₁ mice (4.5 month old, n=20) weretreated twice per week by intraperitoneal injection for 18 weeks usingeach of the following dosing regimes: 5 or 15 mg/kg murine surrogate(MW≅=160 KDa); 4.68 or 14 mg/kg anti-mB7RP1 (MW≅=150 KDa); 1.88 or 5.6mg/kg αBAFF (MW≅=64 KDa); a combination of αBAFF (1.88 or 5.6 mg/kg) andanti-mB7RP1 (4.68 or 14 mg/kg); murine IgG1 (15 mg/kg; an isotypecontrol); or phosphate buffered saline (PBS) (a negative control).Proteinuria was measured in urine using ALBUSTIX® (Bayer, Elkhart, Ind.)every two weeks starting at 5 months of age. The incidence ofproteinuria was expressed as the percentage of mice with urine proteinat a concentration of at least 300 mg/dl in two consecutivemeasurements. Serum anti-dsDNA IgG level was measured by ELISA. Scoringfor kidney disease of all mice was performed by examination of kidneytissue samples for eight different kinds of lesions, that is, glomerularcapillary proliferation, mesangial cell hyperplasia, increased mesangialmatrix, glomerular tuft adhesion, parietal epithelial hyperplasia,interstitial nephritis, tubular dilation/protein casts, and tubularatrophy/interstitial fibrosis. Each type of lesion was given a severityscore from 0 to 5, for a maximum possible score of 32. The scores ofeach group of mice were averaged. Survival was monitored.

At 12 months of age, none of the mice treated with the murine surrogateat either dose level developed proteinuria. In contrast, 100% of micetreated with murine IgG1 or PBS at both dose levels tested exhibitedproteinuria. FIGS. 8A and 9B. About 60% and 35% of mice treated with thelower dose levels of anti-mB7RP1 and αBAFF, respectively, and about 50%and 25% of mice treated with the higher dose levels of anti-mB7RP1 andαBAFF, respectively, developed proteinuria. FIGS. 8A and 9B. Inaddition, the murine surrogate treatment at both dose levels resulted ina significant reduction in serum levels of anti-dsDNA IgG as compared tothe negative control treated with muIgG1. FIGS. 8B and 9A. Thebispecific treatment also significantly improved survival compared withthe mIgG and PBS control groups. Data not shown. However, no cleardifference in survival was observed between the bispecific vs. thesingle agent treatments at the time of experiment termination.

Kidneys from all treated mice, including mice deceased before the end ofstudy, were collected for histology scoring for severity of kidneydisease. The groups of mice treated with αBAFF, the combination of αBAFFplus anti-mB7RP1, or the murine surrogate had significantly lower scoresfor kidney disease as compared to the control group treated with mIgG1.FIG. 10. Groups treated with the surrogate bispecific or the combinationalso showed a trend towards reduced kidney pathology compared to thesingle agent treatment groups, a result that correlates well with theproteinuria results described above. Compare FIG. 10 to FIGS. 8A and 9B.In summary, dual inhibition of BAFF and B7RP1 by the murine surrogate orby a combination treatment with αBAFF plus anti-mB7RP1 was moreeffective than inhibition of only BAFF (αBAFF) or only B7RP1(anti-mB7RP1) in preventing disease onset and progression in the NZB/WF₁ lupus model.

To determine whether inhibition of both BAFF and B7RP1 could effectivelyinhibit the symptoms of murine collagen-induced arthritis, the followingexperiment was done. Male DBA mice were immunized with 100 μg of bovinetype II collagen emulsified in 2× Complete Freund's adjuvant (CFA) onday 0 and boosted with bovine type II collagen in Incomplete Freund'sAdjuvant (IFA) on day 21. Mice were treated with one of the testsubstances twice per week during the 41 week course of the studystarting on day 0. The percentage of mice in each group exhibitingarthritis symptoms and an average arthritic score for each group wasassessed at each time point. Arthritis scores were determined byexamining each limb and assigning a score from 0-3 for each limb, withhigher scores for more swollen and/or inflamed limbs. So the maximumtotal arthritis score is 12. A mouse was counted as having arthritis ifit had an arthritis score of at least 1 in any limb.

Results are shown in FIG. 11. These data indicate that the combinationof αBAFF and anti-mB7RP1 (filled circles connected by solid lines) wasmuch more effective at suppressing arthritis symptoms than either αBAFF(open circles connected by solid lines) or anti-mB7RP1 (filled circlesconnected by dashed lines) alone. The negative control groups treatedwith mIgG (filled squares connected by solid lines) or PBS (filledsquares connected by dashed lines) had the highest percent incidence ofarthritis and highest arthritic scores. These results suggest thatinhibiting both BAFF and B7RP1, as opposed to inhibiting only one ofthese pathways, could be an effective treatment of an autoimmune and/orinflammatory arthritic condition such as rheumatoid arthritis.

What is claimed is:
 1. A nucleic acid encoding a bispecific protein thatcan bind B7RP1 and BAFF, wherein the bispecific protein comprises (a) apolypeptide comprising the amino acid sequence of SEQ ID NO:17 or SEQ IDNO:18; and (b) an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO:19.
 2. A vector comprising the nucleic acid ofclaim
 1. 3. A host cell comprising one or more nucleic acid(s) encodinga bispecific protein that can bind B7RP1 and BAFF, wherein thebispecific protein comprises (a) a polypeptide comprising the amino acidsequence of SEQ ID NO:17 or SEQ ID NO:18; and (b) an immunoglobulinlight chain comprising the amino acid sequence of SEQ ID NO:19; and/orone or more vector(s) that comprise(s) the one or more nucleic acid(s).4. A method for making a bispecific protein that can bind B7RP1 andBAFF, wherein the method comprises: (a) culturing a host cell containingone or more nucleic acid(s) encoding a bispecific protein comprising (i)a polypeptide comprising the amino acid sequence of SEQ ID NO:17 or SEQID NO:18 and (ii) an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO:19 under conditions such that the one or morenucleic acid(s) is (are) expressed, and (b) recovering the bispecificprotein from the cell mass or the culture medium.
 5. The method of claim4, wherein the host cell is a mammalian cell.
 6. The method of claim 3,wherein the host cell is a CHO cell.